231 research outputs found
Aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis
Hutchinson-Gilford progeria syndrome is a rare genetic disorder that mimics certain aspects of aging prematurely. Recent work has revealed that mutations in the lamin A gene are a cause of the disease. We show here that cellular aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by a period of hyperproliferation and terminates with a large increase in the rate of apoptosis. The occurrence of cells with abnormal nuclear morphology reported by others is shown to be a result of cell division since the fraction of these abnormalities increases with cellular age. Similarly, the proportion of cells with an abnormal or absent A-type lamina increases with age. These data provide clues as to the cellular basis for premature aging in HGPS and support the view that cellular senescence and tissue homeostasis are important factors in the normal aging process
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Internal iamin structures within G1 nuclei of human dermal fibroblasts
The nuclear lamina is a mesh-like network of fibres subjacent
to the inner nuclear membrane that is believed
to be involved in the specific spatial reorganisation of
chromatin after mitosis. To determine how the lamina
might be involved in chromatin reorganisation, we have
performed indirect immunofluorescence studies on quiescent
and proliferating human dermal fibroblasts
(HDF). Two monoclonal antibodies recognising human
lamins A and C and three different fixation methods
were employed. In indirect immunofluorescence studies,
cultures of quiescent cells displayed a uniform perinuclear
distribution of the antibodies. In proliferating cultures
two distinct populations of cells were observed:
one population displayed a typical perinuclear antibody
distribution, while the second population displayed an
unusual pattern consisting of a series of spots and fibres
within the nucleus. By inducing cell-cycle synchrony in
cultures we were able to determine that the unusual
internal distribution of the lamin antibodies was
restricted to cells in G1. Optical sectioning and 3-D
reconstruction of the lamina structures in G1 nuclei was
performed with a confocal laser scanning microscope
(CLSM). This revealed that the internal lamin structures
consisted of small foci and fibres proliferating
throughout the nucleus. These structures were shown to
be closely associated with areas of condensed chromatin
but not nuclear membrane. As cells progress towards S
phase the internal lamin foci disappear
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Chromosome positioning is largely unaffected in lymphoblastoid cell lines containing emerin or A-type lamin mutations
Gene-poor human chromosomes are reproducibly found at the nuclear periphery in proliferating cells.
There are a number of inner nuclear envelope proteins that may have roles in chromosome location and
anchorage, e.g. emerin and A-type lamins. In the last decade, a number of diseases associated with tissue
degeneration and premature aging have been linked with mutations in lamin A or emerin. These are
termed laminopathies, withmutations in emerin causing Emery–Dreifuss muscular dystrophy. Despite highly
aberrant nuclear distributions of A-type lamins and emerin in lymphoblastoid cell lines derived from patients
with emerin or lamin A mutations, little or no change in chromosome location was detected
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Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication
The major residual structure that remains associated with
the nuclear envelope following extraction of isolated nuclei
or oocyte germinal vesicles with non-ionic detergents,
nucleases and high salt is the lamina (Fawcett, 1966;
Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). The
nuclear lamina is composed of intermediate filament
proteins, termed lamins (Gerace and Blobel, 1980; Shelton
et al., 1980), which polymerise to form a basket-weave
lattice of fibrils, which covers the entire inner surface of the
nuclear envelope and interlinks nuclear pores (Aebi et al.,
1986; Stewart and Whytock, 1988; Goldberg and Allen,
1992). At mitosis, the nuclear envelope and the lamina both
break down to allow chromosome segregation. As a consequence,
each structure has to be rebuilt during anaphase
and telophase, allowing cells an opportunity to reposition
chromosomes (Heslop-Harrison and Bennett, 1990) and to
reorganise looped chromatin domains (Franke, 1974;
Franke et al., 1981; Hochstrasser et al., 1986), which may
in turn control the use of subsets of genes. Because of the
position that it occupies, its dynamics during mitosis and
the fact that it is an essential component of proliferating
cells, the lamina has been assigned a number of putative
roles both in nuclear metabolism and in nuclear envelope
assembly (Burke and Gerace, 1986; Nigg, 1989). However,
to date there is little clear cut evidence that satisfactorily
explains the function of the lamina in relation to its
structure. In this Commentary we will describe some of the
recent work that addresses this problem and attempt to
provide a unified model for the role of lamins in nuclear
envelope assembly and for the lamina in the initiation of
DNA replication
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Identification of an interchromosomal compartment by polymerization of nuclear-targeted vimentin
A number of structural and functional subnuclear compartments have been described, including regions
exclusive of chromosomes previously hypothesized to form
a reactive nuclear space. We have now explored this
accessible nuclear space and interchromosomal
nucleoplasmic domains experimentally using Xenopus
vimentin engineered to contain a nuclear localization signal
(NLS-vimentin). In stably transfected human cells
incubated at 37°C, the NLS-vimentin formed a restricted
number of intranuclear speckles. At 28°C, the optimal
temperature for assembly of the amphibian protein, NLSvimentin
progressively extended with time out from the
speckles into strictly orientated intranuclear filamentous
arrays. This enabled us to observe the development of a
system of interconnecting channel-like areas. Quantitative
analysis based on 3-D imaging microscopy revealed that
these arrays were localized almost exclusively outside of
chromosome territories. During mitosis the filaments
disassembled and dispersed throughout the cytoplasm,
while in anaphase-telophase the vimentin was recruited
back into the nucleus and reassembled into filaments at the
chromosome surfaces, in distributions virtually identical to
those observed in the previous interphase. The filaments
also colocalized with specific nuclear RNAs, coiled bodies
and PML bodies, all situated outside of chromosome
territories, thereby interlinking these structures. This
strongly implies that these nuclear entities coexist in the
same interconnected nuclear compartment. The
assembling NLS-vimentin is restricted to and can be used
to delineate, at least in part, the formerly proposed
reticular interchromosomal domain compartment (ICD).
The properties of NLS-vimentin make it an excellent tool
for performing structural and functional studies on this
compartment
Rapid chromosome territory relocation by nuclear motor activity in response to serum removal in primary human fibroblasts
This article has been made available through the Brunel Open Access Publishing Fund.Background: Radial chromosome positioning in interphase nuclei is nonrandom and
can alter according to developmental, differentiation, proliferation, or disease status.
However, it is not yet clear when and how chromosome repositioning is elicited.
Results: By investigating the positioning of all human chromosomes in primary
fibroblasts that have left the proliferative cell cycle, we have demonstrated that in
cells made quiescent by reversible growth arrest, chromosome positioning is altered
considerably. We found that with the removal of serum from the culture medium,
chromosome repositioning took less than 15 minutes, required energy and was
inhibited by drugs affecting the polymerization of myosin and actin. We also
observed that when cells became quiescent, the nuclear distribution of nuclear myosin
1ß was dramatically different from that in proliferating cells. If we suppressed the
expression of nuclear myosin 1ß by using RNA-interference procedures, the
movement of chromosomes after 15 minutes in low serum was inhibited. When high
serum was restored to the serum-starved cultures, chromosome repositioning was
evident only after 24 to 36 hours, and this coincided with a return to a proliferating
distribution of nuclear myosin 1ß.
Conclusions: These findings demonstrate that genome organization in interphase
nuclei is altered considerably when cells leave the proliferative cell cycle and that
repositioning of chromosomes relies on efficient functioning of an active nuclear
motor complex that contains nuclear myosin 1ß.Brunel Open Access Publishing Fun
Non-random chromosome positioning in mammalian sperm nuclei, with migration of the sex chromosomes during late spermatogenesis
Chromosomes are highly organized and
compartmentalized in cell nuclei. The analysis of their
position is a powerful way to monitor genome organization
in different cell types and states. Evidence suggests that the
organization of the genome could be functionally important
for influencing different cellular and developmental
processes, particularly at early stages of development (i.e.
fertilization and the consequent entry of the sperm nucleus
into the egg). The position of chromosomes in the sperm
nucleus might be crucial, because their location could
determine the time at which particular chromatin domains
are decondensed and remodelled, allowing some epigenetic
level of control or influence over subsequent paternal gene
expression in the embryo. Here, we analyse genome
organization by chromosome position in mammalian
sperm nuclei from three breeds of pig, as a model species.
We have mapped the preferential position of all
chromosomes (bar one) in sperm nuclei in two dimensions
and have established that the sex chromosomes are the
most internally localized chromosomes in mature sperm.
The distribution of two autosomes and chromosomes X and
Y in sperm heads was compared in primary and secondary
spermatocytes and spermatids in porcine testes. The sex
chromosomes were found at the nuclear edge in primary
spermatocytes, which correlates with the known position of
the XY body and their position in somatic cells, whereas,
in spermatids, the sex chromosomes were much more
centrally located, mirroring the position of these
chromosomes in ejaculated spermatozoa. This study
reveals the temporal repositioning of chromosome
territories in spermatogenesis
Farnesyltransferase inhibitor treatment restores chromosome territory positions and active chromosome dynamics in Hutchinson-Gilford progeria syndrome cells
Copyright @ 2011 Mehta et al.; licensee BioMed Central Ltd. This article has been made available through the Brunel Open Access Publishing Fund.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a premature ageing syndrome that affects children leading to premature death, usually from heart infarction or strokes, making this syndrome similar to normative ageing. HGPS is commonly caused by a mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. Progerin cannot be processed as wild-type pre-lamin A and remains farnesylated, leading to its aberrant behavior during interphase and mitosis. Farnesyltransferase inhibitors prevent the accumulation of farnesylated progerin, producing a less toxic protein. RESULTS: We have found that in proliferating fibroblasts derived from HGPS patients the nuclear location of interphase chromosomes differs from control proliferating cells and mimics that of control quiescent fibroblasts, with smaller chromosomes toward the nuclear interior and larger chromosomes toward the nuclear periphery. For this study we have treated HGPS fibroblasts with farnesyltransferase inhibitors and analyzed the nuclear location of individual chromosome territories. We have found that after exposure to farnesyltransferase inhibitors mis-localized chromosome territories were restored to a nuclear position akin to chromosomes in proliferating control cells. Furthermore, not only has this treatment afforded chromosomes to be repositioned but has also restored the machinery that controls their rapid movement upon serum removal. This machinery contains nuclear myosin 1β, whose distribution is also restored after farnesyltransferase inhibitor treatment of HGPS cells. CONCLUSIONS: This study not only progresses the understanding of genome behavior in HGPS cells but demonstrates that interphase chromosome movement requires processed lamin A.This work was funded by an ORSAS award and the Brunel Progeria Research Fund
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The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts
The sites of nascent DNA synthesis were compared
with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells
were compared with the distribution of PCNA by
indirect immunofluorescence microscopy and laser
scanning confocal microscopy (LSCM). In common
with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe
coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly,
nuclei which had been isolated from quiescent
cultures of HDF were innoculated into cell-free
extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated
at multiple discrete spots some 15min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.Brunel Open Access Publishing Fun
Schistosomes and snails: A molecular encounter
Copyright © 2014 Knight, Arican-Goktas, Ittiprasert, Odoemelam, Miller and Bridger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Copyright © 2014 Knight, Arican-Goktas, Ittiprasert, Odoemelam, Miller and Bridger. Biomphalaria glabrata snails play an integral role in the transmission of Schistosoma mansoni, the causative agent for human schistosomiasis in the Western hemisphere. For the past two decades, tremendous advances have been made in research aimed at elucidating the molecular basis of the snail/parasite interaction. The growing concern that there is no vaccine to prevent schistosomiasis and only one effective drug in existence provides the impetus to develop new control strategies based on eliminating schistosomes at the snail-stage of the life cycle. To elucidate why a given snail is not always compatible to each and every schistosome it encounters, B. glabrata that are either resistant or susceptible to a given strain of S. mansoni have been employed to track molecular mechanisms governing the snail/schistosome relationship. With such snails, genetic markers for resistance and susceptibility were identified. Additionally, differential gene expression studies have led to the identification of genes that underlie these phenotypes. Lately, the role of schistosomes in mediating non-random relocation of gene loci has been identified for the first time, making B. glabrata a model organism where chromatin regulation by changes in nuclear architecture, known as spatial epigenetics, orchestrated by a major human parasite can now be investigated. This review will highlight the progress that has been made in using molecular approaches to describe snail/schistosome compatibility issues. Uncovering the signaling networks triggered by schistosomes that provide the impulse to turn genes on and off in the snail host, thereby controlling the outcome of infection, could also yield new insights into anti-parasite mechanism(s) that operate in the human host as well.NIH-NIAID and the Malacological Society of London
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